REVIEW
The advantages of nanobodies and the prospects these offer
in cancer treatment
Peter van Doorn
Universiteit Utrecht, Thesis for the master Molecular and Cellular Life Sciences
Abstract: VHH fragments, also called nanobodies, derived from heavy-chain only antibodies (HCAb)
originally found in camels, are the so far smallest known antibody fragments that are still capable
of antigen binding with high specificity. These nanobodies have properties that make them superior in many ways compared to conventional antibodies. They are therefore being used in an increasing number of varying applications ranging from uses in consumer products because of their
low costs of manufacturing to the inhibition of enzymes because of their unique binding capabilities and small size. In therapeutics, however, nanobodies receive special attention in the treatment
and diagnosis of cancer. Properties like better tissue penetration make them suitable for the
treatment of solid tumors and their monomeric nature for instance opens up other strategies in
cancer therapeutics. In this review an overview is presented of several advantages that nanobodies
have together with some of the applications that make use of these. The emphasis of the possible
uses of nanobodies, however, is on their use in cancer therapy. The mentioned advantages of
nanobodies are described in relation to possible new and improved applications they offer in regard to cancer treatment.
Introduction
Since the approval in therapeutics of the first
antibody in 1986 antibodies have been an
important focus in drug development. This
first drug started with the conventional Immunoglobulin G (IgG), the most abundant type
of antibody. IgG are tetramers of 150 kDa
consisting of two identical heavy chains that
are each 50 kDa and 2 identical light chains of
25 kDa. These chains are linked to each other
by disulfide bonds [1]. For antigen binding
these immunoglobulins require a variable
domain at the N-terminal end of the light
chain and one at the heavy chain [1]. High
production costs and other downsides like
their large size and relative instability however
led research to smaller antibody derived
fragments. The first ones for instance were
the Fab fragments (~55 kDa) that consist only
of the light and heavy chain, without the 2
constant domains CH2 and CH3 (Figure 1a).
Later Fv fragments consisting only of the variable heavy (VH : 13.6 kDa) and variable light
(VL : 12.4 kDa) chain were made (Figure 1a)
[2]. These Fv fragments however were found
to dissociate upon dilution and had to be
linked together by a single-chain (sc), resulting
in the scFv fragments (~28 kDa) (Figure 1a)[3].
Antibodies consisting of only the VH domain
Figure 1. (A) Schematic representation of a conventional
antibody and its antibody derived fragments. (B) Schematic representation of a heavy-chain only antibody and the
VHH derived from these. HCAb (Heavy-chain only antibody), CH (constant heavy domain), CL (constant light
domain), VH (variable heavy domain), VL (variable light
domain), VHH (variable heavy domain from HCAb).
1
(~12-15 kDa) from normal IgG have also been
tested [4]. And although these even smaller
VH domains do retain some antigen specificity
(even light chain only fragments were found
to retain activity [5]) the removal of the VL
domain from a Fv fragment exposed a too
large hydrophobic surface. This resulted in the
VH molecules to aggregate and consequently
their affinity to drop even further than that of
scFv which already had a lower affinity than
conventional IgG [6]. Fortunately many of the
problems the antibody derived fragments had
were solved when it was found that designing
smaller antibodies was not something that
had to be done in the lab since it was already
occurring in nature.
ly removed by the light chain. BiP however
retains the heavy chain by a stable interaction
with the CH1 domain [11].
Differences between VH and VHH
Because VH from mice or humans and VHH
from Camelidae are all originating from vertebrates they show a not unsuspected high degree of similarity between their genes. Why it
then is that these VHH are functional were VH
fragments are not, or at least in a much smaller degree, lies in certain structural adaptations
that VHH underwent. VHH are different from
normal VH because of the substitution of a
number of amino acids that make the before
mentioned hydrophobic surface of the VH
more hydrophilic [12]. The positive effect of
these mutations in HCAbs was corroborated
by Davies & Riechmann when they ‘camelised’
human antibody fragments by mutating 3 of
these amino acids and showed a significantly
The structure of HCAb
Although the structure of antibodies is highly
conserved among mammals several variations
have been found that lack the VL chain and
only have the VH chain. These antibodies have
for instance been seen in the heavy chain disease [7] and in cartilaginous fish like sharks [8]
wherein naturally occuring heavy-chain only
antibodies (HCAb) exist. The third type of
HCAb is the one receiving the most attention
and was first found by Hamers-Casterman et
al. in camels [9]. The VH fragment of these
HCAb found in Camelidae (Figure 2) are also
called VHH to distinguish them from the normal VH that originate from the classical antibodies. When these VHH are recombinantly
obtained they are also called “nanobody” because of their size. The difference between
VHH and VH originates from a missing CH1,
one of the constant domains of the heavy
chain, in HCAb [1] (Figure 1b). Besides a heavy
chain with a molecular mass that is about 10
to 12 kDa lower this results in a heavy chain
lacking the domain required for binding with
the light chain. Without this domain the heavy
chain binds directly to the hinge region and no
light chain is present [10]. Although this CH1
domain is also encoded by the heavy chain
gene in HCAb, like in normal antibodies, these
HCAb later miss this domain because of a
point mutation in the splice site behind the 3’
end of the CH1 exon [10]. Deletion of the CH1
domain for functional HCAb seems to be crucial since the chaperone BiP that retains heavy
chains in the endoplasmic reticulum is normal-
Figure 2. X-ray structure of a VHH fragment from a HCAb.
The part of the scaffold that would normally be interacting
with the VL domain in a regular FV is shown in light green.
The CDR1, CDR2 and CDR3 domains are shown in red,
orange and yellow respectively. Between the CDR3 and
CDR1 domain a stick figure is shown to indicate the disulfide bond connecting these two domains. The figure was
adapted from Desmyter et al. 1996 [33].
2
reduced aggregation [13]. Beside these distinct features three other characteristic properties of VHH can be found in their hypervariable complementarity determining regions
(CDR). First, VHH have a typically longer CDR3.
The domain is on average 17 residues long as
opposed to the 12 residues in humans or even
9 in mice [14]. Additionally this CDR3 domain
also has an additional disulfide bond, besides
the conserved intradomain disulphide bond,
that forms a bond with a cysteine in the CDR1
domain [12] (Figure 2). The last feature lies in
the hypervariable CDR1 domain that is extended towards the N-terminal end [15] and
has an additional hypervariable region at residues 27-30 which are important for somatic
mutations and are most likely important for
the VHH in binding to antigens [16]. The combination of these longer CDR regions and the
occurrence of loop conformations not present
in, for instance, human antibodies may provide VHH with a bigger repertoire of antigen
binding [15] or perhaps it might help compensate for the lacking VL binding site.
pared to conventional antibody derived VH
[12], VHH also have a very high stability. A
number of VHH that were incubated for one
week at 37 °C kept 100% of their activity and
others had a still remarkable 80% activity left
[17]. VHH overall were found to be very resistant to heat-induced denaturation when
they were incubated at temperatures of up to
80 °C over a longer period of time [23]. Some
VHH were even capable to still bind antigen
specifically at 90 °C. None of the mouse antibodies that where targeting the same antigen
where active at this temperature [24]. This
higher thermal resistance is most likely due to
the decreased hydrophobocity of VHH since
‘humanizing’ VHH to resemble VH resulted in
a lower heat resistance [25]. On top of that
Dumoulin et al. found that besides thermal
resistance, activity could also be fully restored
after chemical denaturation by high concentrations of guanidinium chloride and urea [26].
The high robustness against denaturing conditions might in the future prove to be useful for
the use of nanobodies outside of therapeutics
in industry and research. Also the refolding
after denaturation of VHH could prove to be
advantageous in large scale protein production when recombinant nanobodies are produced in inclusion bodies and will have to be
diluted and refolded before use. One of the
key features of VHH, that might be of great
use in the future, is the fact that besides their
nanomolar affinity (reviewed by [27]) they
have the capability of binding unique
epitopes. This originates from the adaptations
of their hypervariable regions that had to be
introduced due to the absence of a light chain
and thereby three missing CDRs [12]. The originating distinct loop conformations and longer
CDR3 domain allow VHH to create epitope
binding surface areas that are as large as normal VH-VL pairs have and bind into grooves
and cavities of enzymes (reviewed by [28])
that conventional antibodies because of their
flat or slightly grooved surface cannot reach
[29]. In fact, VHH even seem to prefer to penetrate these active grooves in enzymes [30].
Therefore VHH or nanobodies could for instance also be used as enzyme inhibitors since
these cavities in enzymes can play an important role in their enzymatic activity. This
kind of inhibitory activity has already been
Advantages of VHH
The smallest intact antibody derived fragments, Fv or scFv (Figure 1), that can be generated from the normal antibodies are about
30 kDa. However, recombinant VHH, or nanobodies, can be as small as 15 kDa. Because
these nanobodies only consist of a single chain
they have a short time from gene to protein
when they are recombinantly being expressed. This could be seen by the high levels
of protein when recombinant single domain
VHH were expressed in Escherichia coli [17]
and even higher levels could be seen in Saccharomyces cerevisiae [18]. All of these levels
are at least 10 times higher than that what
could be obtained with scFv constructs [17].
The later mentioned yeast production might
however not be favorable in therapeutics because they cannot modify human glycosylation structures. Furthermore, yeast specific
oligosaccharides are added that result in increased immunogenicity (reviewed by [19]).
Production has however also been proven to
be capable in animal cells [20], plants [21] and
insect cells [22]. Besides the mentioned advantage of the increased solubility as a result
from certain amino acid conformations com3
proven to work in carbonic anhydrase [31] and
lysozyme [32,33]. It was also observed that
nanobodies in ELISA experiments blocked
substrate cleavage which further proved that
these antibodies can have enzyme inhibitory
effects [31]. The property of binding to unconventional epitopes in combination with their
size will allow nanobodies to penetrate tissue
more effectively than conventional antibodies
and bind to epitopes that these antibodies
would not be able to reach nor bind. It has to
be noted however that this small size, although having several advantages, also has its
drawbacks. Molecules that are smaller in size
than approximately 60 kDa (estimated kidney
clearance threshold) have a high renal clearance. A short half-life might however also
prove to be useful or even essential when for
instance nanobodies are used as targeting
mechanism and are coupled to toxic molecules (reviewed by [34]). In this case exposure
to healthy cells must be kept to a minimum
and a long half-life would be a major drawback. However, an increased half-life is preferred in many other applications. The solution to this problem however is relatively
easy; make the nanobody bigger. This has
proven to work by fusing nanobodies to long
lived proteins like albumin [35] or immunoglobulin [36], another antibody targeting one
of these long lived proteins [37] or adding
them to a chemical like polyethylene glycol
[38]. In all cases this greatly increased the halflife of the nanobodies. Although VHH derived
nanobodies seem to be so different from
normal VH, because they have so much more
suitable properties over conventional antibodies, the overall homology between VH and
VHH is quite high. The effective result is that
VHH, unlike for instance mouse antibodies
that can cause an immune response [39],
would be more easily applicable in therapeutics. Although it is possible to combine antibody sequences of humans and mice to reduce the immune response of murine derived
antibodies [40], it is easier to “humanize” VHH
because of their relatively high similarity to
VH. A disadvantage of VHH is however that on
their own they cannot elicit functions as antibody-dependent cytotoxicity or complementdependent cytolysis due to their missing Fc
domains. Bispecific nanobodies however, who
will be discussed later in more detail, offer the
possibility of restoring these functions [41].
Obtaining antigen specific nanobodies
Several techniques can be applied to obtain
antigen specific antibodies. The most common
ones are the direct cloning of the VHH genes
from B-cells obtained from peripheral blood or
lymph nodes [42] and the creation of synthetic
libraries. Both of these have their advantages
and disadvantages.
For the direct cloning of VHH genes
first the immunization of a HCAb producing
animal like a llama or dromedary is needed
(Llamas are often preferred though since they
are easier to keep because of their size and
are easier to immunize [43]). mRNA then has
to be isolated from the blood for cDNA synthesis (Figure 3A-C). This can be amplified by a
single set of primers in a PCR (Figure 3D) since
all VHH belong to the same family and are
encoded by a single exon [15]. These genes
differ between the various B-cells but always
have the same homologous border sequences.
It is important though to select primers that
do not amplify the VH genes that also occur in
Camelidae. Because of the hydrophobic surface of VH that is exposed with the missing VL
fragment, the VH fragments aggregate more
easily, resulting in aspecific binding during the
selection of specific antibodies [12,13,44].
Another possibility is of course to select the
PCR fragments on their size since VHH fragments with their missing CH1 domain (Figure
1b) have shorter fragments. Although there
are multiple techniques that can be used, the
mostly preferred option to select specific antibodies is the use of a phage display (reviewed by [45]). A phage display library can be
constructed by cloning the obtained VHH DNA
fragments in a phage genome (Figure 3E)
where they are fused to one of the genes that
encodes for a coat protein [17]. After transfection (Figure 3E) and assembly of these phages
in a bacterium this will result in the VHH being
expressed on the coat of the phage. This then
allows for panning (binding to an immobilized
antigens) (Figure 3F). After a wash step only
the phages that express a VHH specific for the
immobilized antigen will remain bound (Figure
3F). These wash steps can also be repeated
multiple times with different conditions to
4
select for the highest specificity and stability.
Since the remaining phages still have the genetic material of the expressed VHH they can
then be used to infect bacteria so their DNA
coding for the VHH can be used for subsequent steps (reviewed by [46]). This technique
of selecting VHH is similar as used for conventional antibodies with the drawback that these
consist of 2 variable domains that work together. The VH en VL domain both need to be
amplified with different primer sets and
cloned into constructs. Furthermore the linking of these 2 fragments in a construct is done
randomly since the original combination no
library has to be constructed to find the antigen specific antibody-fragment combination.
VHH therefore have the advantage in this application since they consist of just one variable
domain, reducing the amount of work needed
to obtain antigen specific antibodies.
In synthetic libraries the first steps of
obtaining the cDNA are quite similar (Figure
3A-D). The main difference though is that this
can also be done without prior immunization
of the animal. Non-immune libraries of VHH
have in this way been created [48,49]. With
the same panning and selection as previously
described antibodies against a desired antigen
can be obtained. The downside of this however is that the affinity compared to antibodies
derived from an immunized library is low (in
the micromolar range [50] while this normally
is in the nanomolar range (reviewed by [27])).
These affinities can however be improved by
in vitro affinity maturation (Figure 3D-2). The
antigen binding capabilities of these antibodies are (partially) build artificially in this technique. Random mutations are introduced in
the CDR regions of the antigen with for instance an error prone PCR and splicing and
shuffling of these sequences [51]. By selection
of the desired antigen via panning and then
repeating the mutation steps VHH with increased affinity can be obtained in a way that
mimics the in vivo affinity maturation. By using different selection procedures also VHH
with different physical properties like heat
resistance or solubility and stability [50] can
be obtained. Synthetic libraries have several
advantages over their immunized counterparts. One of these for instance is not having
to purify proteins to immunize animals, that
are costly to keep and maintain. Not to mention the constant ethical debates about using
animals for scientific purposes. More importantly however is that in this way antibodies can be obtained against antigens that
could not be used to immunize animals because they are toxic [52] or pathogenic [51].
Furthermore immune libraries are normally
made for a specific single antigen whereas
synthetic libraries can be applied more readily
to a bigger variety of antigens. The downside
is however the inherent low affinity of antibodies obtained via non-immunized libraries.
Figure 3. Schematic representation of key steps to obtain
specific VHH. (A) Blood is obtained from a HCAb producing
animal. (B) mRNA is isolated and (C) used for cDNA synthesis. (D) primers specific for VHH are used to amplify
VHH coding genes. (D2) When VHH with a higher affinity
are to be created an additional step with in vitro affinity
purification can be performed before (E) cloning the
genes in the phage display vectors and transforming them
to bacteria to create a gene library. When using immunized animals this step is usually not needed. (F) The phages created by the bacteria are then panned on immobilized antigen and washed to obtain those with specific
binding. (G) The remaining phages are then used to transfect bacteria so the DNA coding for the specific VHH can
be used in subsequent steps.
longer exists [47]. Therefore a much larger
5
They therefore need (time consuming) affinity
maturation steps.
er antibodies that can bind these antibodies
mimic the original epitope, the catalytic site of
the enzyme. These are called catalytic antibodies, or abzymes, and can mimick the enzymatic active site of an enzyme [56]. As a
model system nanobodies were used in this
way to create abzymes with alliinase activity
that could, in combination with alliin, suppress
tumor growth in vitro [57].
A perhaps less obvious application,
but also worth mentioning function of nanobodies is in aiding the formation of crystals for
x-ray crystallography. The main bottleneck in
x-ray crystallography is the formation of the
crystals. Although natural partner proteins can
alleviate this problem by stabilizing these proteins these are not always available. Alternative binders like nanobodies with their unique
binding properties offer a solution. Besides
stabilizing complexes [58] and organizing disordered proteins [59] for crystal formation,
the property of nanobodies to bind to active
sites [30] have also allowed the identification
of proteins in an active conformation. Nanobodies were able to lock the β2 adrenergic
receptor in that state by mimicking an agonistic G protein that are normally absent because
of their instability [60]. Other ways in which
nanobodies have been helpful is by stabilizing
flexible multidomain proteins into more rigid
hetero tetramer formations and thereby improving the crystal formation [61]. To illustrate to what extend nanobodies aid the formation of crystals; an EpsI-EpsJ dimer crystal
could be made 20 times faster (15 days instead of 10 months) with the help of nanobodies [62].
Uses for nanobodies
So far nanobodies or VHH have been described to be advantageous because of their
high expression, solubility and stability, high
specificity, epitope binding properties unseen
by conventional antibodies, immunogenicity
and more. Al these additional (and beneficial)
properties on top of the ‘normal’ features of
conventional antibodies open up a whole array of possible uses for antibodies. Because of
these varying features nanobodies have already been suggested for applications in different fields. These range from the possible
use as probes for the measurement of caffeine in hot beverages because of their high
thermal stability [53] to the use as compounds
to increase the antibiotic sensitivity of bacteria [54] because of their enzyme inhibitory
functions [28]. These are however just a few
of the possible ways that exploit the features
of nanobodies.
Unique binding properties
One of the potential uses of nanobodies is in
the use of anti-idiotypic agents [27]. These
type of agents function as a replicate of the
original antigen (originating from e.g. a tumor
associated antigen) and can for instance be
used in a vaccine (reviewed by [55]). The distinct structures of the antigen-binding site of
VHH might make nanobodies ideal candidates
since they could target epitopes that VL-VH
combinations could not. More importantly
however, for these type of vaccinations it is
important that the immune response is targeted to the CDR region and not the scaffold.
Here lies the other advantage of nanobodies.
While their CDR region is so distinct from conventional antibodies VHH do show a high similarity with VH fragments. This should shift the
hosts immune response from the scaffold to
the more important CDR region that is mimicking the antigen. Besides their use as antiidiotypic agents in vaccines anti-idiotypic
nanobodies can also be used for different
purposes. The property of nanobodies to
preferentially bind in the active sites of enzymes [30] makes them ideal for the production of catalytic anti-idiotypic antibodies. Oth-
Stability
Because of their high conformational stability
[26] nanobodies are ideal for oral uptake since
they have the ability to survive the harsh conditions in the stomach and intestines. Proteolytic stable nanobodies could therefore be
selected that were not degraded in vivo and
were able to bind the diarrhea causing Escherichia coli in pigs [63]. Similar results were
obtained with nanobodies that were able to
survive the acidic environment of mice intestine and could still target the diarrhea causing
rotavirus [64]. In a completely different example the high stability of nanobodies was found
6
to be of use in shampoo for the treatment of
dandruff. Nanobodies were able to survive the
surfactants present in the shampoo and retain
their ability to bind to Malassezia furfur, a
dandruff causing fungus [65]. This kind of stability make nanobodies also ideal candidates
as capturing agents in affinity columns [66,67]
and biosensors (reviewed by [68]) to name
just a few of the various possibilities.
er (reviewed by [73]). scFvs are made by linking VH and VL chains together. These linkers,
although they can be improved [74], are susceptible to proteolysis and subsequent aggregation. Therefore, fusing two scFvs together,
what will increase the number of linkers
needed from one to three, is not desirable.
Nanobodies however seem to again have the
ideal properties to improve on this technique
(Figure 4B). The by default monomeric nature
of nanobodies reduces the number of linkers
needed. The creation of bi-specific nanobodies
has therefore already shown to be promising
with solubility and expression levels that are
comparable to the monomeric variants [75]. In
a different experiment the power of bi-specific
nanobodies have been shown by viral targeting that was improved up to a 4000-fold after
the fusion of nanobodies that targeted different epitopes [76]. Other reasons why nanobodies are preferred for this application are
their stability, solubility, ease of production
and naturally their size. Bivalent nanobodies
are still five times smaller than monomeric
intact antibodies, thereby still keeping their
better tissue penetrating capabilities. Multivalent antibodies are not only created by fusing
two different antibodies together though.
Size
Besides the discussed properties like their
unique binding abilities and high stability the
feature that makes nanobodies so supreme in
many other applications is their small size.
Besides properties as increased tissue penetration, reaching epitopes conventional antibodies cannot [69] and even showing promise
of crossing the blood-brain barrier [70], the
small size and consequential simple structure
make nanobodies ideal building blocks to be
fused to other molecules thereby further increasing their capabilities.
As described before [35,36] nanobodies can be made into bivalent constructs with
the goal of increasing their half-life (Figure
4A). Bivalent constructs with conventional
antibodies have however been created before
with different goals, namely in the form of bispecific antibodies [71] (Figure 4B). These
types of antibodies have numerous possible
applications in therapeutics. Multiple epitopes
on an antigen could be targeted at the same
time, for instance, increasing their affinity and
specificity. Other applications could be in the
treatment of cancer if bispecific antibodies
can first bind immune cells with one of their
antibodies and consequently help recruit
these cells to tumor cells with the help of their
other antibody. This type of format is also
called a crosslinking reagent. Bispecific (normal) antibodies with this goal have indeed
been tested and reports claimed better results
than with the monomeric counterparts [72].
Of course this kind of application could also be
used with other types of diseased cells. Where
conventional antibodies however already had
the disadvantage of being big, and thus having
reduced tissues penetrating capabilities, these
multivalent versions have this problem doubled. Bi-specific antibodies have therefore
been made by fusing the smaller scFvs togeth-
Figure 4. Examples of bivalent nanobodies. (A) A VHH
fragment and a long lived protein like albumin for example
can be fused together to obtain nanobodies with a longer
half-life. (B) Other possibilities are the fusion of 2 VHH
fragments, either targeting the same or different epitopes,
to increase their affinity or recruit cells to a desired location for instance. (C) Finally, a commonly used technique is
the fusion of VHH to toxins or drugs like certain enzymes
that are needed at specific locations. With the help of VHH
these can be targeted there.
7
Fusing nanobodies that target the same
epitope together for instance to form dimers
or trimers has already been shown to increase
their potency up to a 4000-fold when going
from a monomer to a trimer by the company
Ablynx, the eponym of the nanobody [77]. As
of late even pentamers have been created
that could increase the binding a 1000 to
10,000 fold for low affinity antibodies and
showed promise in the use of antigen discovery [78,79]. Other applications for these multimers can be found in the use of synthetic
library derived antibodies [49] who often have
a low binding affinity.
Coupling of nanobodies to other molecules to increase their half-life or affinity
have been mentioned (Figure 4A). There are
however more possible uses when it comes to
coupling nanobodies to different molecules.
Drugs, like those used in chemotherapy, might
be very effective in killing their targets. The
targeting on itself however will not always be
as precise. Antibodies on the other hand can
be extremely specific in their targeting, but a
single domain antibody like a VHH on its own
will then again not have the same results as
the drug. The combination of these types of
molecules therefore seems a logical next step
in the creation of bifunctional constructs
(Figure 4C). This approach of combining a selective targeting molecule to a, for that same
target, toxic molecule, is also called a “magic
bullet”. This approach with immunofusions
has been tested before with conventional
antibodies. Already in 1988 Senter et al. fused
antibodies to alkaline phosphatase, an enzyme
able to convert etoposide phosphate into the
antitumor drug etoposide, resulting in an antitumor activity that could not have been
achieved by administering the drug on its own
[80] showing the validity of this type of application. Because these types of antibodies consist of multiple domains that need to be in the
right conformation for proper functioning
fusion to a toxin is therefore less straightforward than with the monomeric nanobodies.
Fusions of nanobodies to toxins have already
been successfully tested in varying applications. In an effort to increase the selectivity of
the in oral care used antimicrobial agent glucose oxidase this drug was fused to VHH targeting Streptococcus mutans. Consequently S.
mutans was found to be more susceptible to
this “magic bullet” than the toxin on its own.
Although the overall protection was still low
the hybrid molecule could selectively target
Streptococcus mutans and locally increase
antimicrobial activity [81]. Besides bacteria
also drug resistant parasites, causing sleeping
sickness, have been successfully targeted by
this approach. Because of the unique features
of nanobodies the cryptic and less immunogenic epitopes of Trypanosoma brucei
rhodesiense could be targeted [82]. Fusing
these VHH with apolipoprotein L-1, a protein
that lyses trypanosome, resulted in clearance
of the parasite [83]. These bivalent constructs
are however not all based on the fusion of a
nanobody to a toxic molecule. Rothbauer et
al. showed that nanobodies are also ideal in
diagnostic tools when fused to a fluorescent
molecule. With a new proof of principle they
showed that these nanobodies, when targeting endogenous proteins, allowed for the tracing of the targeted antigens in living cells [84].
Another example of nanobodies fused to a
non-toxic compound, and that more importantly shows how diverse their application
can be, is seen in the laundry industry where
cellulose targeting VHH have been found to
more efficiently target fragrances to clothes
[85]. This just goes to show that the possible
uses for these multivalent formats are enormous. Since the technique behind the fusion
of nanobodies to other molecules is more or
less the same for all of above mentioned fusions it are the same advantages as in other
nanobody based constructs that make them
suitable for the use in “magic bullets”. Their
monomeric nature for instance makes the
fusion of a bivalent construct genetically more
straightforward. This is more problematic with
regular antibodies because of the multiple
domains that are needed in the right conformation for proper functioning.
This monomeric nature combined with
their small size is also the reason why nanobodies easily fold to their natural conformation when expressed in vivo. Complete
antibodies and also scFv fragments on the
other hand are already more complex than
nanobodies. The disulfide bonds needed between the two scFv fragments for instance do
not always form in vivo depending on the in8
tracellular targeting. scFv targeted to the ER
for instance have their disulfide bonds
formed, but the ones targeted to the cytosol
do not [86]. It is therefore that nanobodies
might be good candidates to be used as intrabodies, antibodies that are expressed intracellular. Intrabodies are a powerful method to
bind and inhibit functions of targeted proteins
in vivo (reviewed by [87]). Their potential was
first seen in Saccharomyces cerevisiae where
cDNA encoding for a light- and heavy-chain
against alcohol dehydrogenase I neutralized
its enzyme activity in vivo [88]. The possible
use for these intrabodies in therapeutics was
then also shown by an increased virus infection protection in transgenic plants expressing
a scFv antibody without assembly requirements against essential viral proteins [89].
These scFv have later also been used in human
cell lines with for instance the inhibition of an
influenza A virus [90]. Although nanobodies
have not been used as intrabodies as often as
scFv have in studies they do already show
great promise. In planta for instance they
were found to fold properly, were able to
target different organelles and most importantly were able to bind to enzymes to
inhibit their function [91]. Nanobody derived
intrabodies were the first to be used in in vivo
studies in mammals and showed proof of
principle by inhibiting the secretion of hepatitis B in mice [92]. Nanobodies expressed intracellular have even shown to have potential in
the treatment of HIV-1. In infected cells nanobodies could bind to Rev, a protein controlling
the expression of HIV proteins, and block the
formation of Rev multimers. This prevented
the production of viral HIV-1 [93]. As mentioned scFv as intrabodies have already been
tested in more varying applications. These
range from a possible use in therapeutics
against multiple sclerosis, certain types of
cancer [94] and the treatment of protein misfolding diseases like prion diseases [95],
parkinsons disease (reviewed by [96]) and
huntington’s disease [97] to different approaches in the treatment of HIV-1 [98] as
opposed to the nanobody variant. The advantages that nanobodies offer over scFv are
however very likely to shift future research
from scFv to nanobodies as they also open up
other strategies for intrabodies in regard to
their unique binding epitopes and enzyme
inhibitory functions. The only drawback here
that has to be overcome, by all intrabodies, is
the transfection of the genetic material in
animals. Namely, these in vivo transfection
efficiencies are still very low. So although intrabodies offer many possible strategies in
treating a variety of diseases the main problem mentioned is not with the nanobodies,
but in getting to the required location. Getting
these nanobodies intracellular might however
be a problem that can be solved with nanobodies themselves. This will be discussed in
more detail in the following chapter.
(Possible) applications for nanobodies in cancer treatment
Conventional antibodies have already been
used in the treatment of different diseases for
several years. As mentioned before however
these antibodies have several downsides, or
lacking features, compared to nanobodies.
The usage of nanobodies as new therapeutic
antibodies is therefore expected to greatly
increase upon the efficiency and possible new
applications compared to conventional antibodies. However, the treatment of diseases
with antibodies requires the targeting of specific markers. Cancer for instance will be a
disease subjected to many drug trials with
nanobodies. This is reflected by the fact that
already over 50% of the conventional antibodies that are in development are meant for
cancer treatment (reviewed by [99]). But what
is a cancer marker, and is there even such a
thing? Some characteristics for a tumor marker would obviously be that they are consistently (over) expressed on tumor cells and ideally not on healthy cells (reviewed by [100]). A
number of these type of markers that allow
for the distinguishing between cancer and
healthy cells are already in use by current
monoclonal antibodies (reviewed by [101]).
Nanobodies have however also proven to be
excellent at tumor targeting with properties
like high specificity, rapid clearance of excess
antibodies and the fact that they are nonimmunogenic [102]. Some of the previously
mentioned markers have therefore also been
targeted by nanobodies. For instance, nanobodies have been tested that target the carcinoembryonic antigen (CEA) [103], epidermal
9
growth factor receptor (EGFR) [104], human
epidermal growth factor receptor-2s (HER2)
[105] and mucine 1 (MUC1) [106] to name just
a few. With the advent of nanobodies however a whole new array of markers might be
exploited that could previously not be targeted. The unique features that nanobodies offer
will likely improve upon existing therapies and
possibly open up whole new possibilities in
the treatment of cancer. Here the advantages
of nanobodies and the possibilities that these
open for varying applications are discussed in
regard to current and future applications for
cancer treatment.
The most important thing before antibodies can target their specific antigens is of
course getting to that antigen. Here the advantage of the nanobodies small size comes
into play. Tissue penetration is an obstacle
were many conventional antibodies have to
deal with. And although 85% of all cancers are
solid, only a relative small percentage of cancer drugs based on antibodies are targeting
these solid tumors. This is probably due to the
increased difficulty of targeting these types of
tissue (reviewed by [107]). The higher resistance to these drugs originates from the
extracellular matrix of tumors that is more
difficult to pass through because of extracellular matrix proteins [108]. Also the slower
blood flow in tumors complicates their targeting [109]. Therefore to effectively penetrate
tumor tissue the size of molecules is of great
importance. This can be seen by the diffusion
rate between complete IgG molecules and
scFv fragments. The later can diffuse 12 times
faster than whole IgG antibodies (6 hours vs
30 min) [110]. This higher penetration rate is
however paid for by a shorter retention, also
due to their size. As mentioned before however, this does not have to be a bad thing.
Their small size opens up to a plethora of other beneficial properties besides higher tumor
penetration that can be used in the treatment
of cancer. One of the advantages of having a
good tumor penetration and fast clearance for
instance is of use in tumor markers. These are
necessary for the first step in the treatment of
cancer, namely a rapid and accurate diagnosis.
For this application a long retention is not
necessary and the properties of nanobodies
have already shown promise in the diagnosis
of prostate cancer [42] and breast cancer
[111] for instance.
Monomeric nature of nanobodies
The EGFR is a tumor associated antigen (TAA)
already targeted by many different antibodies
in several studies [112,113]. Nanobodies
against EGFR have now also been studied
[104] because they are easy to identify and
select for in phage display libraries and offer
many advantages for follow up experiments
like fusion to different types of molecules
because of their monovalent nature. The
nanobodies selected in these experiments
were found to block EGF mediated signaling
and thereby inhibit cell proliferation. Besides
offering a new targeting mechanism for EGFR
this study shows that the monomeric nature
of nanobodies allow for the fast development
of antigen specific antibodies [104].
These properties of nanobodies can
also proof to be of use in perhaps less obvious
applications. It has been shown for instance
that certain bacteria like bifidobacterium, clostridium, and salmonella preferably target tumors over healthy tissue to replicate in [114].
This feature could be further exploited by the
use of nanobodies. Since it is already known
that nanobodies can be readily expressed in
vivo in different expression systems [17,18,2022] because of their (simple) monomeric nature and hydrophilicity, expression in these
type of bacteria might also be possible. And
since it is also known that intact nanobodies
can be transported across the outer membrane [115] these nanobodies could be presented on the cell wall to help in the targeting
of tumor cells. After insertion of the bacteria
in the tumor other nanobodies that are expressed and secreted from these bacteria
could aid in killing tumor cells by targeting
certain cancer markers. This might prove to be
beneficial since the nanobodies targeting
these tumor markers are expressed in the
location where they are needed. This might
potentially not only increase their efficacy but
also reduce possible side effects. A similar
technique with cells that target tumors and
express nanobodies has already been tested
recently. This was done with stem cells instead of bacteria. These stem cells could target brain tumors and secrete EGFR targeting
10
nanobodies on the desired location resulting
in a significant reduction of tumor growth
[116]. The proposed targeting of cells by expressing antibodies on their surface is also a
technique that, on its own, has already proven
to work. Cytotoxic T cells have been targeted
to tumors with antibodies that are presented
extracellular. These antibodies, linked to an
extracellular spacer domain and an intracellular signaling domain, are able to activate the T
cell upon binding (reviewed by [117]). This has
normally been done with the use of scFv, the
use of nanobodies might however be preferred. And indeed T cells with nanobodies
instead of scFv to help with their targeting
have already been successfully directed to
tumor cells by MUC1 targeting [118]. All in all
directing cells with the help of nanobodies and
the secretion of functional nanobodies are
strategies that have been proven to work separately. When combined in one cell, as proposed with the bacteria, they might however
have synergistic effects that could benefit
both techniques.
As mentioned the use of intrabodies
shows great potential in inhibition of targeted
proteins. Getting there however might prove
to be more problematic. Nanobodies could be
able to help with both of these points.
Bispecific scFv have been found to be able to
target DNA to tumor cells in the form of adenoviral vectors [119]. As mentioned though,
scFv bivalent constructs have some disadvantages that bivalent nanobodies do not
have because of their monomeric nature.
Therefore by using nanobodies in these constructs instead of scFv the targeting on itself
could be improved. Another method wherein
nanobodies can help in the targeting of genes
to cancer cells is with non-viral vectors. Such
non-viral vectors consisting of polyethylenimine (PEI) and polyethylene glycol (PEG)
for example are very efficient at transfecting
cells but are not very specific in their targeting. Nanobodies however have been found
very useful for this application when covalently linked to the PEG. MUC1 targeting nanobodies in combination with a PEG-PEI conjugate
were in this way able to transfect a plasmid
coding for a killer gene behind the cancer specific MUC1 promoter [120]. This resulted in
cell death in tumor cell lines. Without the
MUC1 promoter this did not happen indicating
that the genetic code was indeed transfected
and the non-viral transfection with PEG and
PEI itself was not the reason for cell death
[120]. Besides transfecting killer genes this
could obviously also be used for the transfection of a gene expressing an intrabody against
an intracellular cancer marker to increase the
specificity. One of these intracellular targets
could for instance be the transactivation domain of the c-Myc gene, a gene that is constitutively expressed in many cancers and regulates cell proliferation among other things
(reviewed by [121]). This domain is already
known to be targetable by scFv antibodies
[122]. These scFv were however not internally
expressed but translocated across the membrane via a fusion to a protein transduction
domain resulting in a cell-permeable antibody.
This strategy however required very high
amounts of fused antibodies [122]. Intracellular expression with nanobodies might improve
upon this. Namely, the fact that intracellular
markers can indeed be targeted by intracellular expressed nanobodies has been shown by
the targeting of the (possible) metastasis
marker hnRNP-K (heterogeneous nuclear ribonucleoprotein K) [123].
Bivalent nanobodies
The monovalent nature of nanobodies does
not only make them suitable for the rapid
search of new antibodies against certain targets, but as mentioned they can also be made
bivalent more easily than for instance scFv.
Bivalent conventional antibodies have already
been proven to be advantageous in the treatment of cancer [72]. But also homo dimeric
nanobodies [104] as well as hetero dimeric
nanobodies targeting adjacent epitopes [124]
have shown to improve binding to EGFR compared to their monovalent versions. Other
types of hetero dimeric nanobodies, or bispecific nanobodies, that target completely
different epitopes have been found to be able
to bind NK-cells and activate interferongamma production. Together with the right
tumor marker these bi-specific nanobodies
show promise of being able to recruit NK-cells
to kill tumor cells [125]. The ease with which
these bivalent constructs can be made could
greatly improve upon the targeting of tumor
11
cells. The production of bivalent nanobodies
will namely be more appealing since the resulting constructs are more soluble and stable
and therefore will show more promise to
eventually end up as a future cancer therapy.
and inhibit enzymes [30]. This property can be
of use in cancer therapy since some enzymes
play key roles in tumor growth like ribonucleotide reductase (reviewed by [128]) and topoisomerase II (reviewed by [129]) which has
already been targeted with antibodies [80].
Although some of these enzymes can also be
targeted by drugs, the use of nanobodies
might offer more possibilities in terms of delivery and fusion constructs. For enzymes that
have not yet been able to be targeted by
drugs the use of nanobodies is more obvious
because of the relative ease to create and find
new nanobodies that can target a desired
antigen. Since some of these enzymes are
however located on the inside of cells this
strategy might have to be combined with gene
therapy like the before mentioned example
with the PEG-PEI conjugate. Instead of the in
that example used killer gene a different gene
coding for an intracellular cancer marker could
be used. This could further increase the therapy’s specificity since nanobodies first need to
select for extracellular cancer markers before
the intracellular targeting nanobodies can do
their job.
Bifunctional nanobodies
As described, nanobodies can be fused to toxic molecules relatively easy compared to their
conventional counterparts. This might be especially useful in cancer treatment since
nanobodies in this way can help increase the
specificity of chemotherapy thereby increasing its efficacy and reducing the side effects by
keeping the exposure to healthy tissue to a
minimum. Also because of the fact that they
have a fast blood clearance nanobodies have
been fused to radio nuclides that emit radiation and result in the destruction of cells.
Nanobodies targeting the human epidermal
growth-factor receptor type 2 have in this way
been fused to the radio-metal 177Lu without
affecting the antigen recognition of the nanobody and having a higher tumor uptake compared to healthy tissue [126]. Similar results
have been obtained by fusing 177Lu to EGFR
targeting nanobodies. Additionally these
nanobodies were also fused to albumin what
resulted in a greatly reduced blood clearance,
but it also vastly improved the tumor uptake
[127]. This goes to show that a consideration
has to be made between fast clearance or
better uptake, depending on the type of application. Other successes in the treatment of
tumor cells with bifunctional nanobodies have
been gained by the fusion of β-lactamase to
carcinoembryonic antigen targeting VHH. The
selective binding of the VHH to tumor cells
resulted in a locally increased toxic drug concentration resulting from the β-lactamase that
converted a nontoxic prodrug to its toxic variant [103]. The authors also reported that a
rapid clearance of the multivalent construct
from non-targeted tissue was seen, without
the need of clearance agents. This again
shows the advantage of the nanobodies small
size.
Opinion and perspectives
Since the discovery of HCAb in camels almost
20 years ago VHH fragments or nanobodies
have been used in a growing number of applications. Mainly their small size and monomeric nature make them superior in many ways
compared to conventional antibodies. Furthermore nanobodies are very stable, easily
expressed and functional in vivo, ideal to create multivalent constructs and can recognize
unique epitopes that the conventional antibodies cannot. And these are just a few of
their advantages. The so far only possible
downside is the potential immunogenicity that
might be elicited when nanobodies are fused
into larger constructs. On their own they seem
to cause no significant immunological reaction
but larger constructs are known to cause a
stronger response. This is something that will
have to be tested in the future since the fusion of nanobodies to various molecules is one
of the features that make them so desirable
for many applications. Most likely this will be
no problem though since the fusion of these
molecules to conventional antibodies would
Unique binding properties
Some of the properties like the longer CDR3
domain give nanobodies the unique binding
properties that allow them to more easily bind
12
result in even larger constructs making nanobodies still the better option.
The low cost of producing nanobodies
opens up possible uses in consumer products
like the already mentioned shampoo and detergent. Their stability allows oral uptake,
making them suitable for instance to be mixed
in feed for animals and their unique binding
properties will allow easier use of antiidiotopic agents, for example. It is however
their simple monomeric nature and size that
will show the true capabilities of nanobodies.
This might especially hold true in cancer diagnosis and therapeutics. As mentioned the
largest part of all cancers consists of solid tumors. The difficult targeting of these tumors
however causes a comparatively low number
of drugs to be designed for this purpose.
Nanobodies have shown to be more capable
in penetrating these tumors. With their antigen binding properties and/or drug delivery
possibilities they will be able to target a wider
variety of (solid) tumors. One of the techniques that can be used in cancer therapeutics
and shows a lot of potential with the use of
nanobodies are intrabodies. The only thing
standing in the way of a real breakthrough in
therapeutics for this type of application is
getting these antibodies inside the cell. Gene
therapy would be the most ideal solution but
still has downsides like low efficiency. But
even in the targeting of genes to specific cells
nanobodies have shown great potential for
the future. This just goes to show the wide
variety of applications nanobodies can be
used in. Their broad spectra of applications is
probably also the reason for the constantly
growing interest in these type of antibodies.
Conventional antibodies have been in use for
a longer period of time, explaining why they
are still more often found in various applications. However, the many different uses
wherein nanobodies have already proven to
be superior to conventional antibodies and
the potential they offer in other applications
make it likely that many future therapeutics
and applications will be based on nanobodies
rather than "normal" antibodies. The prediction is therefore that nanobodies will eventually be preferred in the development of new
applications, thereby gradually expanding the
uses for antibodies and likely replacing the
conventional antibodies.
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